Several assay devices are currently marketed. Typically, sandwich immunoassays are performed on these devices using a capture antibody immobilized on a filter or membrane, either directly or indirectly through trapping of antibody-coated latex particles. The specimen or an extract of it is applied to a device containing the filter or membrane with immobilized capture antibody. These commercial devices utilize capillary action to cause the sample and reagents to flow through the filter or membrane into an absorbent material lying beneath it. A sandwich is eventually formed in which one member is labeled with an appropriate signal-generating substituent, e.g. an enzyme label. Addition of substrate will detect the enzyme label by reacting with the enzyme to produce a colored reaction product. Since the enzyme is attached to a member of a sandwich whose existence requires the presence of the antigen, the colored reaction indicates the presence of the antigen as well. Several disadvantages plague these devices, one of which is the poor sensitivity of the assays. For example, specimens containing as many as 10.sup.11 target molecules/mL are usually needed in order for these assays to measure analytes such as human chorionic gonadotropin. One reason for the poor sensitivity is the difficulty of controlling the rate of capillary flow in the assay device. Reagents are removed from the reaction zone by capillary flow before the reactions are complete. More time for antigen-antibody interaction and other steps in building the sandwich or assaying the enzyme would improve the kinetics and allow the detection of smaller amounts of analyte. Another problem plaguing some assay devices having multilayer structures is the development of background color in an absorbent layer which interferes with discriminating between weak positive results and negative results.
One approach to resolve the difficulty in controlling the capillary flow rate is set forth in U.S. Pat. No. 4,366,241, issued to Tom et al., on Dec. 28, 1982, which discloses a method and apparatus for performing sandwich immunoassays. The apparatus is described as comprising an immunosorbing layer to which a member of an immunological pair is attached, a liquid absorbing member and a bibulous liquid flow-resistant disk. The bibulous liquid flow-resistant disk retards entry of liquid to the absorbent layer so that a uniform flow rate is achieved for liquid flowing through the immunosorbing member. Flow is not restricted to the particular area where the member of an immunological pair is immobilized on the immunosorbing member.
Tom et al. further discloses preventing signal production in a first layer of a multilayer immunosorbing zone by adding an enzyme to an intermediate layer to prevent the transfer of compounds from the detection layer. The example provided in column 13 states that the detector enzyme is present in a first layer and in a third layer. The system is designed so that detection occurs in the third layer. The substrate is not added to the first layer; instead it is generated in situ in the third layer. Another enzyme is present in the layer between the first layer and third layer which prevents any substrate from migrating from the third layer to the first layer so that detection occurs only in the third layer. There is no teaching of reducing background color in an absorbent layer by treating the absorbent layer with an enzyme inhibitor which acts directly on the enzyme.
Both U.S. Pat. No. 4,632,901, issued to Valkirs et al. on Dec. 30, 1986, and U.S. Pat. No. 4,092,115, issued to Rupe et al. on May 30, 1978, relate to devices having a structure located above a filter or membrane which permits sample flow onto a permeable member to which a reagent may be attached.
U.S. Pat. No. 4,623,461, issued to Hossum et al. on Nov. 18, 1986, discloses a transverse flow diagnostic device which can be used to perform a variety of immunoassays. The device is structured so that all fluids and reactants flow outwardly through the filter means from the point of application on to a localized portion of the top surface of the filter to peripheral portions of the filter. The absorbent means located in the peripheral portion of the device exercises a transverse capillary action to pull liquid outwardly from the point of application in the filter plane.
U.S. Pat. No. 4,407,943, issued to Cole et al. on Oct. 4, 1983, discloses immobilization of an antigen or antibody on zein-coated internal and external surfaces of a microporous membrane whose surfaces are rendered immunochemically reactive. It is disclosed that structure having large ratios of surface exposed to the volume of fluid flowing through the membrane are used in an attempt to increase the probability of reactive contact with the surface and reduce reaction time. The incubation time is governed by a technique whereby fluid is applied dropwise to the upper surface of the membrane so that the rate of flow of fluid is governed only by the hydrostatic pressure exerted by the drop as it rests on top of the membrane. An absorbent layer is not employed to exert a capillary action to pull the reagents therethrough.
U.S. Pat. No. 4,246,339, issued to Cole et al. on Jan. 20, 1981, discloses a multilayered device for testing liquid samples for the presence of predetermined components. The test device comprises telescoping top and base members which define a liquid reservoir, the top member has one or more test wells which open to the periphery of a microporous membrane. The microporous membrane has a co-reactant immobilized to its internal and external surfaces, which co-reactant is capable of reacting with the analyte. When a resilient means is depressed, the membrane contacts an absorbent member capable, by blotting or capillary action, of absorbing all the liquid passed through the test wells. Unless there is an absorbent layer and a means for bringing it into contact with the lower surface of the membranes, liquid will not pass through the membranes. When the device is not depressed no liquid will pass through the microporous membrane. The absorbent layer comprises a surface layer which is substantially non-wettable, i.e., for normal aqueous solutions it is comparable to the inner lining of diapers, and a substantially wettable layer.
U.S. Pat. No. 3,888,629, issued to Bagshawe on Jun. 10, 1975, discloses a reaction cell, suitable for use in radioimmunoassay, comprising a container having a matrix pad and a support for the matrix pad allowing liquid to flow therethrough. The matrix comprises a porous absorptive material which can bind analyte. The desired chemical reaction can occur within the matrix. Rapid filtration is induced by employing a porous cellular material substantially in contact with the matrix.
U.S. Pat. No. 3,723,064, issued to Liotta on Mar. 27, 1973, discloses a layered testing device to quantitatively determine the concentration of a substance. The device consists of approximately four layers: a first porous layer impregnated with request system, adjacent to the first layer is a membrane having a plurality of regions with different permeabilities to the fluid being tested, adjacent to the second layer is a porous transmission layer followed by a display strip. Selective permeability here results from chemical reaction with the analyte or size selection due to pore size of membrane layer.
Literature describing a Millipore Immunozyme Toxoplasma Antibody Test, No. PB 847 (1979) discloses a cassette having a microporous immunosorbent, porous hydrophobic layer and an absorbent blotter. When the cassette top is depressed the liquid therein flows through the immunosorbent into the blotter. Thus, this device utilizes physical separation to prevent any background color which may form in the absorbent layer from interfering with the detection of results in the upper layer.